Nucleosides are widespread in the living world as building blocks of nucleic acids. They occur as ribonucleosides in ribonucleic acids (RNA) and as deoxyribonucleosides in deoxyribonucleic acids (DNA).
Naturally occurring nucleosides are usually composed of a sugar moiety (ribose or deoxyribose) and an aglyconic heterocyclic moiety. These so-called nucleobases are usually adenine, guanine, cytosine and thymine or uracil.
In addition nucleosides have been found in natural materials that are not components of nucleic acids such as e.g. isoguanosine or 1-methyl-isoguanosine. They often have interesting pharmacological properties.
The object of the present invention was to provide 2xe2x80x2-deoxy-isoguanosines, isosteric derivatives and isoguanosine derivatives as well as their phosphorus compounds. Oligodeoxynucleotides or DNA fragments which contain the compounds according to the invention are suitable for inhibiting the expression of viral genes in biological systems.
The invention concerns compounds of the general formulae I to IV (cf. FIGS. 1 and 2 for graphic formulae) in which
R1=hydrogen or a protecting group, PO3H2, P2O6H3, P3O9H4 or the corresponding alpha, beta and gamma thiophosphates provided that R1 does not denote P3O9H4 in formula III;
R2=hydrogen, hydroxy, phosphoramidite, methylphosphonate, H-phosphonate, a reporter or intercalator group;
R3=hydrogen or a protecting group;
W or/and Z=hydrogen, halogen, xe2x80x94NHxe2x80x94(CH2)nNH2(n=2-12), xe2x80x94Rxe2x80x94CH2xe2x80x94COOH(R=alkylenyl C1-C8), a reporter or intercalator group.
The invention also concerns compounds of the general formula V in which
R1=equals hydrogen or a protecting group, PO3H2, P2O6H3, P3O9H4 or the corresponding alpha, beta or gamma thiophosphates;
R2=equals hydrogen, hydroxy, phosphoramidite, H-phosphonate, methylphosphonate, a reporter or intercalator group;
R3=hydrogen or a protecting group;
R4=hydroxy;
Z=hydrogen, halogen, xe2x80x94NHxe2x80x94(CH2)nNH2(n=2-12), xe2x80x94Rxe2x80x94CH2xe2x80x94COOH(R=alkylenyl with C1-C8), a reporter or intercalator group
provided that R1, R3 and Z are not simultaneously hydrogen or R1 is not simultaneously P2O6H3 when R3 and Z are hydrogen.
The compounds according to the invention of formulae I-IV are produced by either starting with compounds of the general formulae a or b in which
Rxe2x80x2=Rxe2x80x3 and represents an acyl protecting group such as e.g. p-toluoyl or benzoyl, this is reacted with aroyl isocyanates e.g. benzoyl isocyanate and the desired isoguanosine is isolated,
or compounds of the general formulae VI to IX in which W, Z, R1, R2 and R3 have the above-mentioned meanings,
and Hal represents chlorine, bromine or iodine
are converted photochemically by irradiation into the 2xe2x80x2-deoxyisoguanosines.
Compounds of the general formula V are produced analogously in which case one starts with compounds of formulae a, b, VI to IX which contain a xcex2-D-ribo-furanosyl residue instead of the xcex2-D-erythro-pentofuranosyl residue.
A particularly preferred process for the production of the compounds according to the invention is to persilylate deoxyguanosine or guanosine and their derivatives with hexamethyldisilazane and trimethyl-chlorosilazane. Subsequently the 2,6-diaminonucleoside is produced using ammonia and tris(trimethyl)silyl-triflate and selectively deaminated in the 2 position using nitrite to the isoguanosine or deoxyguanosine.
The further derivatization is carried out according to methods well known to a person skilled in the art.
The heterocyclic moiety in the 2xe2x80x2-deoxyisoguanosines and isoguanosines according to the invention can be preferably replaced by the corresponding isosters 7-deaza-isoguanine and 7-deaza-8-aza-isoguanine in which case these bases can in addition contain further substituents on the C-7 of the 7-deaza- or 7-deaza-8-aza-isoguanine and on the C-8 of isoguanine. Such substituents can for example be reporter groups as described below.
Hydrogen, halogen, NHxe2x80x94(CH2)nNH2, Rxe2x80x94CH2COOH, a reporter or intercalator group are particularly preferred.
A diamino group can be introduced at position W and/or Z by halogenating a compound in which W and/or Z is hydrogen (for example with bromine water) and introducing a diamino group in the bromide by nucleophilic substitution. A diaminopentyl or diaminohexyl group is particularly preferably introduced. The desired compound can be prepared from the mixture of amino compounds by chromatographic purification.
A carboxyl group can be introduced by reacting the corresponding halogenated compound with methyllithium and preparing methyl bromide with halogen. This compound is reacted with aminocarboxylic acids (e.g. amino-caproic acid) to form the final product.
The reporter or intercalator groups are preferably coupled in their activated form (e.g. hydroxysuccinimide ester) to the amino or carboxyl group of the compounds according to the invention.
All suitable end groups known to a person skilled in the art can be present on the 3xe2x80x2 and 5xe2x80x2 end of the sugar moiety of the compounds according to the invention. Hydrogen, monophosphate, diphosphate or triphosphate, a reporter group or intercalator group are preferred for the 3xe2x80x2 end and for the 5xe2x80x2 end. A reporter group within the meaning of the invention is understood as a hapten such as biotin or digoxigenin or a fluorescent dye. Suitable intercalator groups are described by Hxc3xa9lxc3xa8ne, C. in xe2x80x9cAntisense RNA and DNA, Curr. Commun. Mol. Biol.; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1987xe2x80x9d and are preferably phenanthroline, acridine, actinomycin or its chromophore or heavy metal complexing agents such as EDTA. Groups which result in a cross-linking of nucleic acids such as e.g. psoralen are also advantageous.
Cyclic phosphoric acid diesters (3xe2x80x2, 5xe2x80x2-cyclophosphates) can be produced from the compounds according to the invention by water liberation between the 3xe2x80x2-OH and 5xe2x80x2-OH of the sugar moiety.
The production of phosphoramidites, H-phosphonates and p-methylphosphoramidites from isoguanosines is carried out analogously to the production of the corresponding deoxyisoguanosine derivatives in which case the 2xe2x80x2-OH group is preferably protected by a triisopropylsilyl group.
The invention in addition concerns 2xe2x80x2-deoxyisoguanosines, isoguanosines and 7-deaza-8-aza-isoguanosine-3xe2x80x2-phosphoramidites, -3xe2x80x2-H-phosphonates and xe2x80x94P-methyl-phosphoramidites protected by bases and sugars. These compounds are suitable as nucleotide building blocks for the production of oligonucleotides.
The nucleotide building blocks according to the invention preferably contain protecting groups on the heterocyclic bases as well as on the 5xe2x80x2-OH and/or 2xe2x80x2-OH groups of ribose.
Amino-protecting groups such as e.g. benzoyl, formamidine, isobutyryl or diphenoxyacetyl groups are preferably used as a protecting group on the heterocyclic bases.
The 5xe2x80x2 or 2xe2x80x2-OH protecting group of the sugar moiety is preferably a triphenylmethyl, monomethoxytrityl, dimethoxytrityl, t-butyl-dimethylsilyl, t-butyldiphenylsilyl, t-butyl-methoxyphenylsilyl or pixyl group
3xe2x80x2-O-(2-cyanoethyl)-N,N-diisopropyl-aminophosphanes and 3xe2x80x2-O-methyl-N,N-diisopropylamino-phosphanes are preferred as phosphoramidites. The H-phosphonates are preferably used as salts.
The production of the monomeric nucleotide building blocks is carried out according to methods familiar to a person skilled in the art such as those described by Gait, M. J. in xe2x80x9cOligonucleotide Synthesis, A Practical Approachxe2x80x9d, IRL Press, Ltd. (1984).
The compounds according to the invention of the general formulae I to IX can be incorporated by DNA or RNA polymerases into oligonucleotides or newly synthesized DNA or RNA in the form of their respective 5xe2x80x2-triphosphates or xcex1-, xcex2- or xcex3-thiotriphosphates.
In a particularly preferred embodiment, these nucleotide building blocks according to the invention are labelled with 32P or 35S.
The invention therefore also concerns a process for the production of oligonucleotides and polynucleotides which contain the compounds according to the invention as building blocks. Such processes can be of a chemical as well as of an enzymatic nature.
The chemical synthesis of oligonucleotides is carried out according to methods known to a person skilled in the art as described for example in Gait, M. J., loc. cit. or in Narang, S. A., xe2x80x9cSynthesis and Application of DNA and RNAxe2x80x9d, Academic Press, 1987.
The production of the oligonucleotides according to the invention is carried out in a known manner, for example according to the phosphate triester, the phosphite triester or H-phosphonate method in a homogeneous phase or on a support. The two latter methods are preferably used whereby the synthesis is usually carried out using automated synthesizers. The support materials in this case consists of inorganic (controlled pore glass, Fractosil) or organic polymeric material (e.g. polystyrene) known to a person skilled in the art.
The invention therefore also concerns a process for the production of oligonucleotides consisting of 6 to 100 nucleotide building blocks according to the oligonucleotide synthesis process in which, depending on the sequence design, appropriate 2xe2x80x2-deoxy-isoguanosines or isoguanosines according to the invention are used wherein a starting nucleoside is bound to a solid support and subsequently the desired oligonucleotide is assembled by stepwise coupling using appropriately activated monomeric nucleotide building blocks, if desired trivalent phosphorus is oxidized to pentavalent phosphorus during or after the synthesis, the oligonucleotide is cleaved from the support with a first base, heterocyclic protecting groups are cleaved with a second base, the 5xe2x80x2 protecting group is cleaved with an acid and the oligonucleotide is purified if desired. The purification is preferably carried out by reverse phase or anion exchange HPLC. This is usually followed by a desalting, for example by dialysis.
In addition the oligonucleotides according to the invention can also be produced enzymatically using polymerases. Such processes are known to a person skilled in the art under the terms xe2x80x9cin vitro transcriptionxe2x80x9d, xe2x80x9cnick translationxe2x80x9d [Rigby et al., J. Mol. Biol. 113, 237 (1977)] and xe2x80x9crandom primingxe2x80x9d [Feinberg, A. P. and Vogelstein, B., Anal. Biochem. 137, 266 (1984)].
In this process 2xe2x80x2-deoxynucleoside-5xe2x80x2-triphosphates or isoguanosine-5xe2x80x2-triphosphates are basically incorporated by polymerases using a single-stranded template nucleic acid and a starter molecule (primer/promoter) into a newly synthesized second strand that is complementary to the bases of the first strand. The use of the nucleoside-5xe2x80x2-triphosphates according to the invention and their corresponding substituted derivatives enables suitable signal groups or reporter groups such as haptens or fluorophores to be incorporated for example into nucleic acids. Such techniques are widely used nowadays for example in the form of non-radioactive labelling of biomolecules.
The invention in addition concerns the use of oligonucleotides in which guanosine or deoxyguanosine building blocks have been replaced completely or partially by the deoxyisoguanosine or isoguanosine building blocks according to the invention and which are composed of 6 to 100 nucleotide building blocks for the production of a pharmaceutical agent with antiviral activity.
Oligonucleotides containing 2xe2x80x2-deoxyisoguanosine form duplex and triplex structures and also aggregates with themselves as well as with other conventional and modified oligonucleotides. In the case of 2xe2x80x2-deoxyisoguanosine this results in manifold base pairing patterns which differ from those of 2xe2x80x2-deoxyguanosine and in which the oligonucleotide strands can be arranged either in parallel or antiparallel.
a) Aggregate Structures
During the preparation of 2xe2x80x2-deoxyisoguanosine it is noticed that it forms gels like 2xe2x80x2-deoxyguanosine and crystallizes with extreme difficulty. Aggregates have been demonstrated by gel electrophoresis for oligonucleotides containing guanine [66] which comply with the structural proposal of Zimmerman [67].
In this structure all N7 atoms are bound up in a Hoogsteen base pairing. The (iGd)4 structure differs considerably from the (Gd)4 configuration since in this case two N7 atoms and two N3 atoms act in addition to the 0 atoms as proton acceptors. As a result 2xe2x80x2-deoxyisoguanosine is able to form a dimeric structure in which one molecule is present as a 1H tautomer and the other as a 3H tautomer.
An interaction via N-7 is not possible in the case of 7-deazapurine derivatives such as 7-deaza-2xe2x80x2-deoxyisoguanosine and 7-deaza-8-aza-2xe2x80x2-deoxyisoguanosine. Hence no aggregate formation is observed with these compounds.
b) Watson-Crick Duplex Structures
With regard to the complexation of single-stranded DNA or RNA, oligonucleotides containing iGd are able to form duplex structures. In this process parallel and antiparallel configurations of the strands are possible. Due to the altered pattern of substituents on the base and the possible parallel duplex structures, an increased stability towards nucleases would be expected.
c) Triplex Structures of DNA Duplexes with Oligonucleotides Containing iGd 
Oligonucleotides which contain 2xe2x80x2-deoxyisoguanosine as monomeric building blocks can form triplex structures with d(AT) duplexes as well as with d(GC) duplexes. In both cases these structures can form in a neutral medium; a protonation is not necessary. As a result the complexation can be achieved under physiological conditions.
The structures discussed above apply in principle to oligoribonucleotides and oligodeoxyribonucleotides.
[66] J. Kim, Ch. Cheong and P. B. Moore, xe2x80x98Tetramerization of an RNA oligonucleotide containing a GGGG sequencexe2x80x99, Nature 1991, 351, 331.
[67] S. B. Zimmerman, xe2x80x98X-ray Study by Fiber Diffraction Methods of a Self-aggregate of guanosine-5xe2x80x2-phosphates with the same Helical Parameters as Poly(rG)xe2x80x99, J. Mol. Biol. 1976, 106, 663.
The oligonucleotides according to the invention and their salts can be used as medicines e.g. in the form of pharmaceutical preparations which can be administered orally e.g. in the form of tablets, coated tablets, hard or soft gelatin capsules, solutions, emulsions or suspensions. They can also be administered rectally e.g. in the form of suppositories or parenterally e.g. in the form of injection solutions. These compounds can be processed in therapeutically inert organic and inorganic carriers for the production of pharmaceutical preparations. Examples of such carriers for tablets, coated tablets and hard gelatin capsules are lactose, maize starch or derivatives thereof, talcum, stearic acid or salts thereof. Suitable carriers for the production of solutions are water, polyols, sucrose, inverted sugar and glucose. Suitable carriers for injection solutions are water, alcohols, polyols, glycerol and vegetable oils. Suitable carriers for suppositories are vegetable and hardened oils, waxes, fats and semi-liquid polyols.
The pharmaceutical preparations can also contain preservatives, solvents, stabilizers, wetting agents, emulsifiers, sweeteners, dyes, flavourings, salts to change the osmotic pressure, buffers, coating agents or antioxidants as well as if desired other therapeutically-active substances.